Method for reducing the number of unwanted molecules in blood

ABSTRACT

The present invention provides a method, and resulting product, for the removal of unwanted molecules from a host&#39;s blood using a one-step procedure. The unwanted molecules may be anti-A blood protein and/or anti-B blood protein antibodies that would otherwise cause host rejection of transplanted organs or tissues from a source having a different ABO blood type. The unwanted molecules may also be excess antibodies, or virions, present in a diseased host.

This application is a Continuation of Ser. No. 09/566,510, filed May 8,2000, now abandoned which is incorporated herein by reference.

FIELD OF INVENTION

This invention relates to technology for reducing the presence ofunwanted molecules, including those related to disease states and thoseinvolved in rejection of transplanted organs and tissue, a host's blood.In particular, the invention discloses a method and a system forreducing the presence of unwanted molecules, such as anti-A and anti-Bantibodies, by a one step removal process. It also discloses bloodsubstantially of unwanted antibodies, antigens, and the like.

BACKGROUND OF THE INVENTION

Traditionally, organ or tissue transplantation requires ABO blood typecompatibility in order to prevent graft rejection. Normally, the host'sblood contains circulating antibodies against foreign blood typeantigens. Transplantation across these ABO blood groups leads tohyperacute rejection of the graft within the first 24 hours (Kuby J:Immunology. New York, W.H. Freeman and Company, 1997). Circulatingantibodies bind to blood antigens present in red blood cells, epithelialcells and endothelial cells found in the graft organ or tissue. Theseantibody-antigen complexes activate the complement system of the host,resulting in infiltration of neutrophils into the graft organ or tissue.The neutrophils release lytic enzymes that destroy the graft endothelialcells, providing a surface of injured tissue to which platelets canadhere. Massive blood clots form within the capillaries, and this wholeinflammatory reaction prevents vascularization.

Current treatments to reduce rejection include administering a regimenof immunosuppressant drugs before and after the transplantation surgery.Studies have been performed on methods that remove antibodies specificto ABO antigens. These methods have also shown beneficial effects inreducing hyperacute rejection of the transplanted organ or tissue. Thesemethods are important because they may lead to a method which will relaxthe requirement of donor/recipient ABO compatibility, which in turn cangreatly expand both the living donor and cadaver organ or tissue pools.

Current techniques to remove the ABO antibodies include plasma exchangecombined with intravenous administration of soluble ABO antigens(Alexandre G P J, et al., Neth J Med, 28:231-234, 1985); separatingplasma from the whole blood by either centrifugation or doublefiltration plasmapheresis (DFPP) followed by immunoadsorption usingconcentrated red blood cells (Slapak M, et al., Transplantation 31:4-7,1981); and DFPP followed by column immunoadsorption of anti-A and Bantibody using A and B antigen bound to silica beads (Tanabe K, et al.,Transplantation Proceedings, 27(1) 1020-1023, 1995).

These prior art methods have serious problems which have prevented theiradoption as the standard of care. First, there is the risk of infection.Because plasma exchange by centrifugation requires replacement by plasmaprotein solution, risk of viral transmission is present. Moreover, thesetechniques described above involve first separation of plasma from wholeblood then an additional procedure to remove ABO antibodies from theplasma. Separated plasma can then be stripped of pre-existing anti-A andB antibodies by immunoadsorption with ABO antigens linked to silicabeads on a column.

A study on renal transplantation has shown that ABO-incompatible graftedpatients who received one or two sessions of DFPP and three or foursessions of column immunoadsorption showed no significant difference insurvival rates when compared to patients who received an ABO compatiblegraft (Tanabe, supra). Additionally, one case has been reported in whichhyperacute rejection following accidental ABO-incompatible renaltransplant was reversed using plasmapheresis followed byimmunoadsorption with red blood cells (Slapak, supra).

SUMMARY OF THE INVENTION

This invention provides a method and system for reduction of a host'srejection of a non-autologous organ or tissue transplant caused by thepresence of foreign antigens in and on the organ or tissue. This isaccomplished by providing a method for one-step removal of antibodies inthe host's blood that are directed to the foreign antigens. For example,cross-ABO rejection can be eliminated by removing anti-A and/or anti-Bantibodies, in one step, from the host's blood. This is done by movingblood extracted from the host along a pathway, which is optionallysemi-permeable, having antigen specific to the antibodies, such asantigens that bind to anti-A and anti-B antibodies, attached to thepathway, and returning the blood to the host internal circulation.

In another embodiment, this invention provides a method for removing, inone step, excess antibodies, such as are present in certain diseasestates, from a host's blood by moving the blood extracted from the hoston a pathway, optionally semi-permeable, having antigens oranti-antibodies specific to the unwanted antibodies immobilized in thepathway, and returning the blood to the host's internal circulation.

In yet another embodiment, unwanted antigen is removed from a host'sblood in one step by moving the blood extracted from the host on apathway, optionally semi-permeable, having antibodies specific to theantigen immobilized in the pathway, and returning the blood to thehost's internal circulation.

In another embodiment, this invention provides blood that issubstantially free of undesired molecules, such as anti-A and anti-Bantibodies, wherein A and B are blood type antigens. The undesiredmolecules may also be antibodies associated with a disease statecomprising an excess of antibodies in the blood, virions, and otherundesired antigens.

In the preferred practice of the invention, a hollow fiber has attachedA and B blood type antigens that are capable of sequestering theantibodies specific to A and B antigens from the flowing blood. Inanother preferred practice of the invention, the hollow fiber withattached antigen has semi-permeable pores that allow dialysis orplasmapheresis of the blood to occur at the same time. In a furtherpreferred practice of the invention, the hollow fiber is coupled to aplurality of perpendicular membranes having attached antigen.Alternatively, this plurality of membranes can also be longitudinallyplaced inside and along the length of the fiber. In the most preferredembodiment, the antigens are attached to the wall of the hollow fiber.In further practice of the invention, the hollow fiber can be replacedby a flat membrane in a closed container that the blood can flow alongor pass through. In this embodiment, an optional semipermeable membraneis present to divide the flowing blood from a slurry that will inducethe blood components, such as antibodies, to exchange across themembrane.

The invention also provides a method to increase the organ or tissuepools available for transplant by removing in one step, from the host'sblood, antibodies specific to foreign antigens present in thetransplanted organ or tissue

The invention also provides a one-step system for removing antibodies tospecific antigens from blood in one step.

The invention also provides a one-step system for harvesting antibodiesto specific antigens from blood.

In another embodiment, the present invention provides circulating bloodthat is substantially free of unwanted molecules, wherein thesemolecules are capable of binding, either specifically ornon-specifically, to a binding partner capable of being immobilized on apathway. In particular, this invention provides circulating blood thatis substantially free of anti-A blood protein and anti-B blood proteinantibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a longitudinal cross section view of an antibody removalsystem in accordance with a first embodiment of the present invention.

FIG. 2 is a longitudinal perspective section view of an antibody removalsystem in accordance with a second embodiment of the present invention.

FIG. 3 is a longitudinal perspective section view of an antibody removalsystem in accordance with a third embodiment of the present invention.

FIG. 4 is longitudinal perspective section view of an antibody removalsystem in accordance with a fourth embodiment of the present invention.

FIG. 5 is a top perspective view of an antibody removal system inaccordance with a fifth embodiment of the present invention.

FIG. 6 shows the results of an assay using the method of this inventionto remove anti-A and anti-B antibodies from blood.

FIG. 7 shows the results of an assay using the method of this inventionto remove anti-A antibodies from blood, showing the high capacity of theproduct.

FIG. 8 shows the results of an assay using the method of this inventionto remove anti-B antibodies from blood showing the high capacity of theproduct,

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention provides a method and a system for one step removal, fromthe host's blood, of antibody specific to foreign antigens present in atransplanted organ or tissue. This is done by moving blood extractedfrom the host along an enclosed pathway such as a hollow fiber or flatdialyzer comprising bound or immobilized specific antigen, and returningthe blood to the host internal circulation. The blood components aredialyzed across the membrane of the pathway, while at the same timeantibodies are removed from the blood through binding to the immobilizedantigen. The binding can be specific, as when the antigens are chosen tobe the specific binding partners of the antibodies, or nonspecific, aswhen a general binding molecule such as protein A or protein G is usedto bind the antibodies.

The antibodies, along with undesired small molecules (urea, creatinine,ammonia), are thus removed from the host's blood. Additionally, theseantibodies can be collected by releasing them from their bindingpartners.

Expanding on this technique, the invention also provides a means forremoving other unwanted molecules from a host's blood. For example,virions present in the blood due to a viral infection of the host can beremoved by utilizing immobilized antibodies, either monoclonal orpolyclonal, to the virion.

Materials

Enclosed Pathway

The present invention comprises an enclosed pathway that allows the flowof blood and the trapping of one of the binding partners of a bindingpair, such as an antibody and an antigen. The device can be made out ofa variety of substances, including but not limited to nitrocellulose,cellulose, nylon, plastic, rubber, polyacrylamide, agarose,poly(vinylalcohol-co-ethylene), and the like, and combinations thereof.The material is preferably semi-permeable to allow the passage of smallmolecules out of the pathway.

The device can be formed in a variety of shapes, including but notlimited to a flat dialyzer, a semi-permeable membrane, a semi-permeablehollow fiber, a coil, a dialysis membrane, a plasmapheresis filter, andmultiples and combinations thereof.

The preferred embodiment as shown in FIG. 1 uses a semi-permeable hollowfiber 1 for commercial dialysis with the antigen 3 attached to the wall4 of the tubing with or without a linker molecule, for example PEG(polyethylene glycol), connecting one to the other. Use of dialysismembranes with attached antigen allows direct membrane immunoadsorptionof the specific antibody 5 and plasmapheresis to occur at the same time.

Alternatively, other anchors for the immobilized binding partner can beused alone or in combination. For example, the hollow fiber 1 can have aplurality of flat membranes 9 that are placed longitudinally along thefiber length (FIG. 3) or perpendicular to the fiber (FIG. 2). Theantigens 3 which are non-diffusively linked to this plurality ofmembranes 9, sequester the specific antibodies 5 from the blood as theypass along the hollow fiber 1. The membranes 9, preferably hi-fluxmembranes, allow blood cells and components to pass through so that noclogging occurs. The tubes themselves may be dimpled, twisted, orotherwise modified to increase mixing and binding of pathogen andantigen.

FIG. 4 shows another embodiment of the hollow fiber 1 where the antigensare linked to free floating permeable spheres 11 located inbetween theplurality of membranes 9. These spheres are trapped between the hi-fluxmembranes because of their size. The antigens 3 on the spheres 11sequester the specific antibodies 5, thus removing them from the blood.Air or other non-toxic gas may be added at a lower elevation as smallbubbles to further mixing and binding, and then the gas can be removedwith a standard bubble trap at a higher elevation (not shown). Thegas-induced mixing can occur on either the shell side or the tube(lumen) side.

FIG. 5 shows another embodiment of the invention, where the antigens 3are attached to flat semi-permeable membranes 13 of a flat platedialyzer 15 instead of a hollow fiber. Blood plasma (as shown bydownward arrows 17) passes through the membrane by convection but thespecific antibodies are retained at the membrane. The blood travelsalong the pathway, continuously or temporarily interrupted, from left toright in the figure.

Binding Pair

This invention can be used with any binding pair, including but notlimited to an antigen and an antibody, a receptor and ligand, ananti-antibody and an antibody, or binding portions of these molecules.By the term “binding portions” is meant any portion of the molecule thatis capable of binding, either specifically or non-specifically, to apartner molecule so as either to be removed or to remove the bindingpartner from the blood.

In the preferred embodiment of the invention, the ABO blood groupantigens are bound to the lumenal surface to remove their correspondingantibodies from blood. The antigen/antibody binding pair can be reversedwherein the antibody is bound to the luminal surface and the antigen isremoved from the blood. Other antibodies, anti-antibodies, and antigens,such as major histocompatibility complex (MHC) molecules, or parts ofthese molecules, can be used to trap antibodies specific to thesemolecules. The antigen/antibody pair can further be replaced with anymembers of sets of binding pairs that would have specific affinities.Examples are ligands and receptors with some specificity to a pathogen.

Substance A and B antigens can be procured from Dade International inSwitzerland (trade name: Neutr-AB). This mixture of Substance A and Bantigen can be from a variety of natural sources, including but notlimited to cows, pigs, horses and humans. These antigens, in their mostreduced form trisaccharides, can also be made synthetically. A higheraffinity for the antigen will exist when the antigen matches theoriginal antigen to which the antibodies were produced. Likewise, themore purified the antigen is, the stronger the reaction.

The more antigen is present, immobilized directly on the lumenal surfaceor attached by a linking molecule in the enclosed pathway, the morespecific antibody can be removed from the flowing blood. Likewise, thelarger the surface area of the coated membrane, the higher the capacityfor binding the desired antibody. For instance, 100 mg of antigennon-diffusively linked to a hollow fiber can significantly reduce theanti-A and anti-B titers of 300 to 400 ml of blood with from average tohigh titer. FIG. 6 shows the capacity of a modified hollow fiber tosequentially process 100 ml of banked human blood. Titer is determinedby using a standard hemagglutination assay. This shows thatmembrane-bound antigen can specifically remove anti-A and anti-Bantibodies, and that this removal takes place in the first 15 minutes offlow (about 3 passages of the blood over the membrane), regardless oforiginal titer. Alternatively, one antigen type, such as Substance A orB, can be used.

FIG. 7 shows the capacity of filters modified with A antigen for anti-Aantibodies. FIG. 8 shows the same using B antigen for anti-B antibodies.Consecutive samples of blood were passed over the membrane until themembrane was saturated. At this point the titer of antibody in the bloodsamples no longer decreased upon passage over the membrane. The anti-Acoated membrane had a capacity of around 300-400 ml. of average to hightiter blood. The anti-B coated membrane had a capacity of around 600 ml.

Further purification of the standard antigens leads to at least a sixfold increase in capacity to remove anti-A and anti-B antibodies per mgof antigen. Purification is achieved by removing components havingmolecular weight below 12,000 daltons from the commercially availableantigen solution by dialysis. For example, the anti-A antibody capacityof a dialysis filter modified with approximately 40 mg of purifiedantigen reduced the anti-A titer of each of six 150 ml blood samples to2 or below. The standard non-purified antigen-modified filter reducedthe anti-A titer of the first sample from 32 to 8, and caused no titerreduction of the other five samples. The results were similar for theanti-B antibodies. Hence we expect that a dialysis filter modified with100 mg of purified may be able to significantly reduce the anti-A andanti-B titers of 1.8 to 2.4 L of average to high titer blood.

Linking; of Binding. to the Enclosed Pathway

The antigen, antibody, binding pair member, ligand, or binding partsthereof, can be linked to the enclosed pathway by a variety of standardlinking techniques, including but not limited to chemical modifications,covalent bonding, strong ionic or hydrogen bonding, use of a linker,etc. The preferred method uses standard cyanogen bromide (CNBr) linkingwhich starts by treating the enclosed pathway with CNBr followed byincubation of the antigen and the modified pathway. The N-terminus ofthe antigen protein will covalently attach to the CNBr linker. Othercompounds for treating the enclosed pathway include, but are not limitedto, hydrogen peroxide, sodium periodate, epichlorohydrin,1,4-butanedioldiglycidol ether, cyanuric chloride, carbonylkdiimidazole,substituted sulfonyl chloride, or fluoromethyl pyridinium salts, andantigen applied in the same way. Standard chemical linkers such asavidin and biotin can also be used.

Process

Filtration

Filtration of unwanted molecules from blood can be achieved usingstandard kidney dialysis type equipment which removes blood from one armand returns it to the other. Alternatively, any pumping system connectedto the patient at two sites, so as to draw blood from one site andreturn it to the other, will work. The blood is passed through theenclosed pathway having immobilized binding partners. The bindingpartners sequester the unwanted molecules as they move along. Severalpasses of the blood along the pathway might be required to completelyremove the specific unwanted molecules.

The flow rate of blood moving through the pathway must be fast enough toprevent coagulation, yet not so fast as to damage the blood cells.Examples of ranges are from about 10 to about 1000 ml of blood per min.,preferably between about 50 and about 750 ml/min. and most preferablythe flow rate for removal of antibodies using the invention is betweenabout 100 and about 500 ml/min. Heparin can also be added to the bloodto prevent coagulation. Processing of an entire host's blood volume (˜5L) would require approximately 2.5 hours to achieve complete removal ofantibodies or other undesired molecules from the blood.

The flow can be continuous. Alternatively, the flow can be interruptedto increase the interaction of the unwanted molecules with theirimmobilized binding partners. Likewise, the shape of the device havingthe immobilized binding partners can be such that it will encourage someswirling and/or backflow to increase the interaction time between theunwanted molecules and the immobilized binding partners.

Uses

The current invention can be used in reducing organ or tissue transplantrejection by removing specific antibodies against foreign antigens foundin the transplanted organ or tissue and providing circulating bloodsubstantially of these antibodies. The invention can also be used aspart of a quantitative assay for specific antibodies found in the blood.For example, a whole body assay for the titer of anti-A and anti-Bantibodies can be performed. First, the antibodies from the blood can beremoved by the filtration described above. Second, the bound antibodiesare released by competing with free floating antigens or with other verylow ionic strength buffers to prevent binding. Third, releasedantibodies can be titered using a method such as a hemagglutinationassay.

Also, the invention can be used to preparatively purify specificantibodies from the blood without the need to plasmapherese. The stepsare similar to the quantification assay described above.

Additionally, the invention can be used to remove excess amounts ofantibodies present in the blood.

Further, the invention can be used to identify, quantify and/or removeother molecules having binding partners, such as virions or ligands,from the host's blood.

1. A method for reducing the number of unwanted antibodies in asubject's blood, the method comprising: a) removing blood from thesubject; b) passing the blood along an enclosed pathway; wherein saidpathway comprises one or more semi-permeable hollow fibers with one ormore membranes having surfaces positioned substantially perpendicular tothe length of the one or more hollow fibers and antigens specific forthe unwanted antibodies immobilized on the one or more membranes,wherein by passing the blood along the pathway the unwanted antibodiesare sequestered by the immobilized antigens, thereby treating the blood;and c) returning the treated blood to the internal circulation of saidsubject, wherein the returned treated blood has a reduced number ofunwanted antibodies compared to before treatment.
 2. The method of claim1, wherein at least a portion of the enclosed pathway is composed of amaterial selected from the group consisting of nitrocellulose,cellulose, nylon, plastic, rubber, polyacrylamide, agarose, poly(vinylalcohol-co-ethylene), and combinations thereof.
 3. The method of claim1, wherein the one or more semi-permeable hollow fibers are at leastpartially dimpled or twisted to increase mixing of the blood within theone or more hollow fibers, thereby increasing contact of the blood withthe immobilized antigens.
 4. The method of claim 1, wherein the step ofpassing the blood through the enclosed pathway comprises continuous ortemporarily interrupted passage of the blood through the enclosedpathway.
 5. The method of claim 1, wherein antigens specific for theunwanted antibodies are further immobilized on the walls of thesemi-permeable hollow fiber.
 6. The method of claim 1, wherein aplurality of membranes are provided within the semi-permeable hollowfiber, thereby dividing the length of the hollow fiber into three ormore successive sections.
 7. The method of claim 6, wherein the enclosedpathway further comprises permeable carrier spheres trapped withinsuccessive sections defined by two membranes, wherein the diameter ofthe spheres is less than the diameter of the hollow fiber, and whereinantigens specific for the unwanted antibodies are further immobilized onthe spheres.
 8. The method of claim 1, wherein the unwanted antibodiesare antibodies associated with a disease state characterized by anexcess of antibodies present in the blood.
 9. The method of claim 1,wherein the unwanted antibodies are selected from the group consistingof anti-A blood protein antibodies, anti-B blood protein antibodies,anti-protein A antibodies, anti-protein G antibodies and antibodiesagainst major histocompatibility complex molecules.
 10. The method ofclaim 9, wherein the unwanted antibodies are anti-A blood proteinantibodies or anti-B blood protein antibodies.
 11. The method of claim1, wherein the antigens are attached to the one or more membranes by aprocess selected from the group consisting of chemical modification,covalent bonding, strong ionic bonding, hydrogen bonding, and use of alinker.
 12. The method of claim 11, wherein the chemical modification isaccomplished by treatment with a compound selected from the groupconsisting of cyanogen bromide, hydrogen peroxide, sodium periodate,epichlorohydrin, 1,4-butanedioldiglycidol ether, cyanuric chloride,carbonyldiimidazole, substituted sulfonyl chloride and fluoromethylpyridinium salts.
 13. The method of claim 1, wherein the antigens areattached to the one or more membranes by avidin or biotin linkers.
 14. Amethod for reducing the number of unwanted antigens in a subject'sblood, the method comprising: a) removing blood from the subject; b)treating said blood by passing the blood along an enclosed pathway;wherein said pathway comprises one or more semi-permeable hollow fiberswith one or more membranes having surfaces positioned substantiallyperpendicular to the length of the one or more hollow fibers andantibodies specific for the unwanted antigens immobilized on the one ormore membranes, wherein by passing the blood along the pathway theunwanted antigens are sequestered by the immobilized antibodies; and c)returning the treated blood to the internal circulation of said subject,wherein the returned treated blood has a reduced number of unwantedantigens compared to before treatment.
 15. The method of claim 14,wherein at least a portion of the enclosed pathway is composed of amaterial selected from the group consisting of nitrocellulose,cellulose, nylon, plastic, rubber, polyacrylamide, agarose, poly(vinylalcohol-co-ethylene), and combinations thereof.
 16. The method of claim14, wherein the one or more semi-permeable hollow fibers are at leastpartially dimpled or twisted to increase mixing of the blood within theone or more hollow fibers, thereby increasing contact of the blood withthe immobilized antibodies.
 17. The method of claim 14, wherein the stepof passing the blood through the enclosed pathway comprises continuousor temporarily interrupted passage of the blood through the enclosedpathway.
 18. The method of claim 14, wherein antibodies specific for theunwanted antigens are further immobilized on the walls of thesemi-permeable hollow fiber.
 19. The method of claim 14, wherein aplurality of membranes are provided within the semi-permeable hollowfiber, thereby dividing the length of the hollow fiber into three ormore successive sections.
 20. The method of claim 19, wherein theenclosed pathway further comprises permeable carrier spheres trappedwithin successive sections defined by two membranes, wherein thediameter of the spheres is less than the diameter of the hollow fiber,and wherein antibodies specific for the unwanted antigens are furtherimmobilized on the spheres.
 21. The method of claim 14, wherein theantibodies are attached to the one or more membranes by a processselected from the group consisting of chemical modification, covalentbonding, strong ionic bonding, hydrogen bonding, and use of a linker.22. The method of claim 21, wherein the chemical modification isaccomplished by treatment with a compound selected from the groupconsisting of cyanogen bromide, hydrogen peroxide, sodium periodate,epichlorohydrin, 1,4-butanedioldiglycidol ether, cyanuric chloride,carbonyldiimidazole, substituted sulfonyl chloride and fluoromethylpyridinium salts.
 23. The method of claim 14, wherein the antibodies areattached to the one or more membranes by avidin or biotin linkers.